Recent advances in sequencing technologies enable the identification of specific mutations in individual tumors, raising the possibility for developing targeted therapeutics for specific tumors. Functional genomics has proven to be a powerful approach for uncovering the underlying drivers of human biological and disease processes. CRISPR-Cas9 and shRNA libraries provide effective screening tools to knockout or knockdown protein-coding genes. Targeting specific oncogenic alterations and pathways in tumor cells has been found to be highly effective for treatment of some cancers including HER2 amplified breast cancer and acute promyelocytic leukemia. However, for many common mutations including activating RAS and loss of TP53, the approach of directly targeting the oncogenic alteration or pathway has proven difficult. Moreover, many diseases and biological phenotypes are caused by gene overexpression or abnormal elevation of gene activity. Therefore, it is highly desirable to utilize forward genetic screens to interrogate the human genome for synthetic lethal interactions in tumor cells with oncogenic mutations. While loss-of-function screens on cancer cells using shRNA libraries have been successfully applied to identify synthetic lethal targets, genome-wide gain-of-function screens for negatively selected genes are lacking.
Therefore, a need exists in the art for improved methods to identify negatively selected genes, especially in the case of common oncogenic alterations that lead to cancer.